rev:January 2, 1997

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ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of researchd Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.


STAINING OF DNA-SYNTHESIZING CELLS WITH BU-FITC


PRODUCT CODE : RDI-CBL 413    $438.00/100T vial  $375.00/vial 3 or more


A) INCORPORATION OF 5'-BROMO-2'-DEOXYURIDINE (CBL 187) INTO DNA


5'-Bromo-2'deoxyuridine (BU) is incorporated into the DNA of S-phase (DNA-synthesizing) cells. With CBL 187 the proportion of cells in the S-phase of the cell cycle can be easily identified, because this antibody is specific for BU-substituted DNA.


BU is simply added to the culture medium at a final concentration of 10-20 µM together with 2'-deoxycytidine (20-50µM). Short pulses (i.e. brief presence of BU in culture medium) of 10 min or less can be detected. However, for routine work pulses of 1-3 h are recommended.


B) DIRECT IMMUNOFLUORESCENCE STAINING


a) Monolayer cells


1. Wash cells, grown on slides or cover slips, twice with PBS.


2. Fix cells with cold methanol for 20 min at -20oC (at this stage cells may be kept for 1 month at -200C).


3. Pre-wet dry cover slips by a short incubation in PBS.


4. Denature DNA to its single-stranded form by two consecutive treatments with 4 M HCl for 15 min each.


5. Wash 3 times with PBS.


6. Incubate with the ready to use anti-BU-FITC-conjugate (30 min, room temperature humidity chamber).


7. Wash twice with PBS.


8. Mount dry samples with standard mounting medium and evaluate the results by fluorescence microscopy.


b) Suspension cells


1. Wash cells twice with PBS (250 g, 7 min).


2. Resuspend cells in 3 vol PBS (0oC) and fix cells by adding 9 vol methanol (-20oC) whilst mixing the cell suspension. Incubate for 20 min.

3. Denature cells by adding one equal volume of 4 M HCl to fixed cell suspension (20 min. room temperature).


4. Pellet cells and resuspend the pellet in 4 M HCl, incubate for 15 min.


5. Carefully remove HCl by three washes with PBS (5000 g, 7 min).


6. Resuspend cell pellet in ready to use staining solution (ca. 25µl/106 cells).


7. Wash twice with PBS.


8. Mount dry samples with standard mounting medium and evaluate the results by fluorescence microscopy.


RDI Divison of researchd Industries Intl

San Jose, 95123 CA Snell ave 658

USA

phone 408-780-0908 or (201) 584-7093

EMAIL:margaret@cellular-research.com

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